Little is known about absorption or metabolism of oxysterols. We compared labeled cholesterol to labeled oxysterols (7(-hydroxycholesterol, 7(-hydroxycholesterol, and 7-ketocholesterol) prepared from 4-14C-cholesterol, [26,26,26,27,27,27--2-H6]cholesterol, and [23,24,25,26,27-13C5]cholesterol. Gastrointestinal absorption of oxysterols in rats was 91.5(0.3% compared to 75(1.1% for cholesterol (p<0.001). When injected intravenously and followed by GC/mass spectrometry, 7(-hydroxycholesterol was cleared at 23 times the rate of cholesterol. After 5 min only 1.2(0.2% of 7(-hydroxycholesterol remained in plasma whereas 28.0(1.7% of cholesterol and 40.0(2.5% of a triglyceride emulsion injected simultaneously remained. 14C-7(-hydroxycholesterol injected intravenously was also rapidly cleared from plasma, widely distributed in tissues, and extensively metabolized. The fractional rate of uptake of radio-labeled oxysterols by cultured endothelial cells was 15.7 times that of choles terol (p<0.001) and the fractional rate of efflux was 3.4 times that of cholesterol (p<0.001). Oxysterols traversed endothelial cell monolayers 4.3 times faster than cholesterol (p<0.001). Fractional oxysterol transport across endothelial cell layers was increased 62. Oxysterols were metabolized to more polar metabolites during endothelial cell transit. These oxysterol properties provide a mechanism for enhancing cholesterol transport through tissues and preventing accumulation of oxidized cholesterol.